Project Summary In the past decade, there has been increasing appreciation for an association between the gut microbiota and numerous cardiovascular phenotypes. One of the most prominent has been the link between gut microbial metabolism of trimethylamine (TMA) moieties from dietary sources (choline, phosphatidylcholine, L-carnitine, etc.) and CVD. Gut microbes metabolize dietary choline to release TMA via the action of cutC TMA lyase. TMA is absorbed and then oxidized by hepatic flavin monooxygenases (FMO3) to form trimethylamine N-oxide (TMAO). TMAO has been causally linked to atherothrombotic disease in animal models and is predictive of CVD risk in cohort studies. The overall objectives of this R21 proposal are to: 1) determine the feasibility and establish proof-of-concept for the effects of choline bitartrate supplementation on circulating TMAO and endothelial-dependent dilation in middle-aged adults in order to conduct a larger, more comprehensive trial in the future; 2) establish our proficiency in measuring gut microbiota composition and function; and 3) obtain preliminary data for effect size generation. To this end, following a two-week lead-in diet, we will randomize twenty-four middle-aged adults (45-65 yrs) to 4-weeks of choline bitartrate (1000 mg/d) or placebo. Subjects will be provided all of their food with choline and TMA-moiety intake maintained at meal levels intake of the US diet for the duration of the study from our metabolic kitchen to avoid potential confounding through differences in habitual dietary intake of TMA moieties between individuals. Measurements of TMAO concentration by UPLC-MS/MS, flow-mediated dilation using high resolution ultrasound, and gut microbiota composition/function using 16S rRNA pyrosequencing and targeted qt-PCR, respectively will be made before and following each 4-week treatment period. This innovative integrative and translational physiological study will be conducted by an established P.I. and investigative team with extensive experience and a strong record of success performing intervention studies targeting cardiometabolic dysfunction. These studies have significant translational potential as they may advance basic science findings in rodents to humans and provide novel mechanistic insight into observational studies in humans by establishing the effect of dietary choline on endothelial function through its interaction with the host intestinal microbiota. In turn, the gut microbiota may be a key target for therapies that may contribute to the maintenance of a healthy endothelium or treatment of endothelial dysfunction. Importantly, our study also will provide insight into the gut microbiota as important source of inter-individual variability in the increase in TMAO and flow-mediated dilation responses to dietary choline intake. As such, the latter may provide rationale for individualizing nutritional or other interventions that target the gut microbiota as an interface between the food we eat and host physiology (i.e., endothelial function).